CN104946708B - A kind of fermentation medium and fermentation process for producing feldamycin - Google Patents

Abstract

The invention discloses a fermentation medium for fermenting and producing antibiotic fidaxomicin by using D. citrus cysts. The raw material components of the fermentation medium include 5-13% of organic carbon source and 1-3.5% of organic nitrogen sources and inorganic salts, the percentages are mass percentages relative to the total mass of the fermentation medium; among them, the organic carbon sources include maltose and glycerin, and the organic nitrogen sources include cottonseed cake powder, soybean cake powder, polypeptone, gluten powder, and yeast extract One or more of powder and yeast powder, the inorganic salt contains CaCO ₃ and/or MgSO ₄ ·7H ₂ O. The invention also discloses two fermentation methods for fermenting and producing fidaxomycin by using Cystium citrus fingernails, including the steps of: activating the bacteria and inoculating (the two methods are respectively primary inoculation and secondary inoculation), and the obtained seed liquid It can be inoculated and fermented in the aforementioned fermentation medium. The fermentation medium and the fermentation method of the present invention greatly increase the fermentation unit of fidaxomicin and are beneficial to industrial production.

Description

Fermentation medium and fermentation method for producing fidaxomicin

technical field

The invention relates to the technical field of industrial microorganisms, in particular to a fermentation medium and a corresponding fermentation method for fermenting the antibiotic fidaxomicin with D. aurantiacus.

Background technique

Fidaxomicin is an eighteen-membered ring macrolide antibiotic with a unique structure and biological activity. It is mainly used for the treatment of diarrhea caused by Clostridium difficile (Clostridium difficile) infection. Application prospect. At present, the chemical synthesis of fidaxomicin has not been reported, and it is mainly produced by microbial fermentation. Fidaxomycin can be metabolized by Dactylosporangium aurantiacum or strain Actinoplanes deccanesis (ATCC21983). In US Pat. No. 7,507,564, fidaxomicin is metabolized and produced by the subspecies hamdenensis of D. aurantiacus, and the disclosed fermentation unit is only 250 mg/l. Therefore, it is necessary to improve the fermentation environment, develop a fermentation medium and a fermentation method that can increase the yield of fidaxomicin, thereby reducing costs and adapting to industrial production.

Contents of the invention

The technical problem to be solved in the present invention is to provide a fermentation medium for producing the antibiotic fidaxomicin by fermentation of D. The fermentation method can greatly increase the fermentation unit of fidaxomicin and improve production efficiency.

One of the technical solutions adopted by the present invention to solve the above-mentioned technical problems is: a fermentation medium for fermenting the antibiotic fidaxomicin with D. Salt; among them, the organic carbon source includes maltose and glycerin, and the amount of organic carbon source is 5-13%; the organic nitrogen source includes cottonseed cake powder, soybean cake powder, polypeptone, gluten powder, yeast extract powder and yeast powder One or more of them, the amount of organic nitrogen source is 1-3.5%; the inorganic salt contains CaCO ₃ and/or MgSO ₄ ·7H ₂ O; the percentage is the mass percentage relative to the total mass of the fermentation medium.

The organic carbon source may also include various organic carbon sources commonly used in fermentation media.

The amount of the organic carbon source is preferably 8.0-13.0%, more preferably 10.0-13.0%, and the percentage is the mass percentage relative to the total mass of the fermentation medium.

The amount of maltose is preferably 4.0-9.0%, more preferably 6.0-9.0%, and the percentage is the mass percentage relative to the total mass of the fermentation medium.

The amount of glycerol is preferably 1.0-4.0%, more preferably 2.0-4.0%, and the percentage is the mass percentage relative to the total mass of the fermentation medium.

The organic nitrogen source preferably contains cottonseed cake powder, soybean cake powder and yeast powder, more preferably contains 1.0-2.0% cottonseed cake powder, 1.0-2.0% soybean cake powder and 1.0-2.0% yeast powder, The percentage is the mass percentage relative to the total mass of the fermentation medium.

The organic nitrogen source is preferably one of 2.0% cottonseed cake powder, 2.0% soybean cake powder, 2.0% polypeptone, 2.0% gluten powder, 2.0% yeast powder or 2.0% yeast extract powder, and the percentage is The mass percentage relative to the total mass of the fermentation medium.

The amount of the inorganic salt is preferably 0.2-0.6%, more preferably 0.4-0.6%, and the percentage is the mass percentage relative to the total mass of the fermentation medium.

Said inorganic salt preferably comprises CaCO ₃ and MgSO ₄ ·7H ₂ O. Among them, the amount of CaCO ₃ is preferably 0.3-0.5%, more preferably 0.35-0.45%, most preferably 0.4%; the amount of MgSO ₄ ·7H ₂ O is preferably 0.1-0.4%, more preferably 0.15-0.25%, most preferably Preferably 0.2%; the percentage is the mass percentage relative to the total mass of the fermentation medium.

The fermentation medium of the present invention, like the conventional fermentation medium, preferably may also contain various inorganic salts of conventional types and dosages in the art other than CaCO ₃ and MgSO ₄ ·7H ₂ O.

The fermentation medium of the present invention is the same as the conventional fermentation medium, and preferably can also contain conventional types and amounts of antifoaming agents in the art. Among them, the defoaming agent is preferably foam enemy, and the amount of foam enemy is preferably 0.05-0.2%, more preferably 0.1%, and the percentage is the mass percentage relative to the total mass of the fermentation medium.

The pH of the fermentation medium of the present invention is preferably 6.8 to 7.5, more preferably 7.1 to 7.4, most preferably 7.3.

In the fermentation medium of the present invention, it is preferable not to contain an adsorption resin.

In the fermentation medium of the present invention, it is preferable not to contain an emulsifier.

The second technical solution adopted by the present invention to solve the above-mentioned technical problems is: a fermentation method for producing the antibiotic fidaxomicin with D. The seed solution is obtained from the seed culture medium, and the seed solution is inoculated in the aforementioned fermentation medium, and fermented and cultured.

In the fermentation method, the inoculum amount of the seed solution is preferably 5-25%, more preferably 5-15%, most preferably 10%, and the percentage is the volume percentage relative to the total volume of the fermentation medium.

In the above fermentation method, the fermentation temperature of the fermentation culture is preferably 25-32°C, more preferably 27-32°C, most preferably 28°C.

In the fermentation method, the fermentation period of the fermentation culture is preferably 8-15 days, more preferably 10-15 days, most preferably 12 days.

In the fermentation method, the container for fermentation culture is preferably a fermentation bottle or a fermenter, more preferably a fermentation bottle.

In the above fermentation method, the fermentation culture is preferably carried out on a shaker, and the rotation speed of the shaker is preferably 100-400 rpm, more preferably 150-300 rpm, most preferably 200 rpm.

In the described fermentation method, the seed liquid can be obtained according to conventional operations in the art, and is preferably produced according to the following steps:

(1) Inoculate fidaxomicin-producing cysts of Citrus tangerinae on the slant medium and activate it. The raw material components of the slant medium include yeast extract powder 0.1-0.4%, malt extract powder 0.1-1%, glucose 0.1-0.4% and 0.1-2.0% agar, the percentage is the mass percentage relative to the total mass of the slant medium, and the pH of the slant medium is 7.0-7.3;

(2) Inoculate the activated colony on the seed medium to obtain the seed solution. The raw material components of the seed medium include glucose 0.05-0.1%, starch 1-2.4%, yeast extract powder 01-0.5%, tryptone 0.1- 0.5%, beef extract 0.1-0.3% and CaCO ₃ 0.1-0.4%, the percentages are mass percentages relative to the total mass of the seed medium, and the pH of the seed medium is 7.0-7.3.

The third technical solution adopted by the present invention to solve the above-mentioned technical problems is: a fermentation method for producing the antibiotic fidaxomicin with D. Obtain the primary seed solution in the seed medium, inoculate the primary seed solution in the seed medium to obtain the secondary seed solution, inoculate the secondary seed solution in the aforementioned fermentation medium, and ferment and cultivate.

In the above fermentation method, the inoculum amount of the primary seed solution is preferably 1-10%, more preferably 3-10%, most preferably 5%, and the percentage is the volume percentage relative to the total volume of the seed medium.

In the fermentation method, the inoculum amount of the secondary seed liquid is preferably 5-25%, more preferably 5-15%, most preferably 10%, and the percentage is the volume percentage relative to the total volume of the fermentation medium.

In the above fermentation method, the fermentation temperature of the fermentation culture is preferably 25-32°C, more preferably 27-32°C, most preferably 28°C.

In the fermentation method, the fermentation period of the fermentation culture is preferably 8-15 days, more preferably 10-15 days, most preferably 12 days.

In the fermentation method, the container for fermentation culture is preferably a fermentation bottle or a fermenter, more preferably a fermenter.

In the fermentation method, the controlled dissolved oxygen in the fermentation culture is preferably 1-30%, more preferably 10-30%, most preferably 20-30%, and the percentage is the volume percentage relative to the total volume of the fermentation medium.

In the above fermentation method, the fermentation culture is preferably carried out on a shaking table, and the rotating speed of the shaking table is preferably 100-400 rpm, more preferably 150-300 rpm, most preferably 200 rpm.

In the described fermentation method, the secondary seed liquid can be obtained according to conventional operations in the art, and is preferably produced according to the following steps:

(1) Inoculate fidaxomicin-producing cysts of Citrus tangerinae on the slant medium and activate it. The raw material components of the slant medium include yeast extract powder 0.1-0.4%, malt extract powder 0.1-1%, glucose 0.1-0.4% and 0.1-2.0% agar, the percentage is the mass percentage relative to the total mass of the slant medium, and the pH of the slant medium is 7.0-7.3;

(2) Inoculate the activated colony on the seed medium to obtain the primary seed liquid. The raw material components of the seed medium include glucose 0.05-0.1%, starch 1-2.4%, yeast extract powder 01-0.5%, tryptone 0.1-0.5%, beef extract 0.1-0.3% and CaCO ₃ 0.1-0.4%, the percentage is the mass percentage relative to the total mass of the seed medium, and the pH of the seed medium is 7.0-7.3;

(3) Inoculate the first-level seed solution on the seed medium to obtain the second-level seed solution. The raw material components of the seed medium include glucose 0.05-0.1%, starch 1-2.4%, yeast extract powder 01-0.5%, tryptone 0.1-0.5%, beef extract 0.1-0.3% and CaCO ₃ 0.1-0.4%, the percentages are mass percentages relative to the total mass of the seed medium, and the pH of the seed medium is 7.0-7.3.

In the two fermentation methods described in the present invention, the fidaxomicin obtained by fermentation culture can be post-treated by the method of conventionally harvesting the fermentation product, preferably the fermentation medium containing the fidaxomicin product is mixed with methanol and then ultrasonicated, After centrifugation, take the supernatant. The methanol volume is preferably 2 to 4 times, most preferably 3 times, the volume of the fermentation medium containing the Fidaxomycin product; the fermentation medium containing the Fidaxomicin product is preferably mixed with methanol to the Methanol is added to the fermentation medium of the daxamycin product; the ultrasonic time is preferably 20-40 minutes, most preferably 30 minutes.

On the basis of conforming to common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The positive and progressive effects of the present invention are: the fermentation medium of the present invention has low cost, greatly increases the fermentation unit of fidaxomicin, improves production efficiency, and is suitable for industrial production.

Detailed ways

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions.

In the following examples, unless otherwise specified, the percentages refer to the mass percentage relative to the total mass of the fermentation medium.

In the following examples, the D. aurantiacus used is the D. aurantiacus strain purchased from the Agricultural Research Service Culture Collection (ARS) with the number NRRL18085.

Example 1

Inoculate a strain of Citrus tangerine finger cysts on the slant medium, cultivate it at 30°C for 4 days and activate it, then inoculate it into the seed medium with a liquid volume of 20ml/250ml, and cultivate it on a rotary shaker at 28°C and 200rmp for 48 hours Get the seed liquid. The seed solution was inoculated into a 250ml fermentation shaker flask with a liquid volume of 30ml at 10% inoculum size (percentage is the volume percentage relative to the total volume of the fermentation medium) for fermentation. The fermentation temperature was 28°C and the fermentation period was 12 days. After the fermentation, three times the volume of methanol was added to the fermentation medium containing the product fidaxomicin, and the supernatant was obtained by centrifugation after ultrasonication for 30 minutes. The yield of fidaxomicin was determined to be 276 mg/l by HPLC.

The raw material components of the slant medium used include 0.4% of yeast extract powder, 1% of malt extract powder, 0.4% of glucose and 2.0% of agar, the pH is adjusted to 7.2, and the percentages are mass percentages relative to the total mass of the slant medium.

The raw material components of the seed medium used include glucose 0.1%, starch 2.4%, yeast extract powder 0.5%, tryptone 0.5%, beef extract 0.3% and CaCO ₃ 0.4%, adjust the pH to 7.3, the percentage is relative to The mass percent of the total mass of the seed medium.

The raw material components of the fermentation medium used include maltose 4.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, Adjust the pH to 7.3.

Example 2

The raw material components of the fermentation medium include maltose 6.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, pH adjusted to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 12 days. Other conditions were the same as those in the example 1. The output of Fidaxomycin reaches 530mg/l.

Example 3

The raw material components of the fermentation medium include maltose 7.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, pH adjusted to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 12 days. Other conditions were the same as those in the example 1. The output of Fidaxomycin reaches 440mg/l.

Example 4

The raw material components of the fermentation medium include maltose 8.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, adjust the pH to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 12 days. Other conditions were the same as those in the example 1. The output of Fidaxomycin reaches 678mg/l.

Example 5

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, pH adjusted to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 12 days. Other conditions were the same as those in the example 1. The output of Fidaxomycin reaches 854mg/l.

Example 6

The raw material components of the fermentation medium include maltose 9.0%, glycerin 1.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, pH adjustment to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 12 days. Other conditions were the same as those in the example 1. The output of Fidaxomycin reaches 384mg/l.

Example 7

The raw material components of the fermentation medium include maltose 9.0%, glycerin 3.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, adjust the pH to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 12 days. Other conditions were the same as those in the example 1. The output of Fidaxomycin reaches 1126mg/l.

Example 8

The raw material components of the fermentation medium include maltose 9.0%, glycerol 4.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, adjusted pH to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 12 days. Other conditions were the same as those in the example 1. The output of Fidaxomycin reaches 1234mg/l.

Example 9

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, cottonseed cake powder 2.0%, CaCO ₃ 0.4% and MgSO ₄ ·7H ₂ O 0.2%, and the pH is adjusted to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 12 days. Other conditions were the same as those in the example 1. The output of Fidaxomycin reaches 811mg/l.

Example 10

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, gluten powder 2.0%, CaCO ₃ 0.4% and MgSO ₄ ·7H ₂ O 0.2%, and the pH is adjusted to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 12 days. Other conditions were the same as those in the example 1. The output of Fidaxomycin reaches 380mg/l.

Example 11

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, polypeptone 2.0%, CaCO ₃ 0.4% and MgSO ₄ ·7H ₂ O 0.2%, and the pH is adjusted to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 12 days. Other conditions were the same as those in the example 1. The output of Fidaxomycin reaches 554mg/l.

Example 12

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, soybean cake powder 2.0%, CaCO ₃ 0.4% and MgSO ₄ ·7H ₂ O 0.2%, and the pH is adjusted to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 12 days. Other conditions were the same as those in the example 1. The output of Fidaxomycin reaches 508mg/l.

Example 13

The raw material components of the fermentation medium include 9.0% maltose, 2.0% glycerin, 2.0% yeast extract powder, 0.4% CaCO ₃ and 0.2% MgSO ₄ ·7H ₂ O, and the pH is adjusted to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 12 days. Other conditions were the same as those in the example 1. The output of Fidaxomycin reaches 394mg/l.

Example 14

The raw material components of the fermentation medium include 9.0% maltose, 2.0% glycerin, 2.0% yeast powder, 0.4% CaCO ₃ and 0.2% MgSO ₄ ·7H ₂ O, and the pH is adjusted to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 12 days. Other conditions were the same as those in the example 1. The output of Fidaxomycin reaches 489mg/l.

Example 15

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, pH adjusted to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 5% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 12 days. Other conditions were the same as the implementation Example 1, the yield of fidaxomicin reached 832mg/l.

Example 16

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, pH adjusted to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 15% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 12 days. Other conditions were the same as the implementation Example 1, the yield of fidaxomicin reached 889mg/l.

Example 17

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, pH adjustment to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 20% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 12 days. Other conditions were the same as the implementation Example 1, the yield of fidaxomicin reached 789mg/l.

Example 18

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, pH adjustment to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 25% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 12 days. Other conditions were the same as the implementation Example 1, the yield of fidaxomicin reached 736mg/l.

Example 19

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, pH adjusted to 6.8.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 12 days. Other conditions were the same as the implementation Example 1, the yield of fidaxomicin reached 816mg/l.

Example 20

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, pH adjusted to 7.5.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 12 days. Other conditions were the same as the implementation Example 1, the yield of fidaxomicin reached 788mg/l.

Example 21

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, pH adjustment to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 25°C, and the fermentation period was 12 days. Other conditions were the same as the implementation Example 1, the yield of fidaxomicin reached 547mg/l.

Example 22

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, pH adjusted to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculum amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 32°C, and the fermentation period was 12 days. Other conditions were the same as the implementation Example 1, the yield of Fidaxomycin reached 768mg/l.

Example 23

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, pH adjustment to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 8 days. Other conditions were the same as the implementation Example 1, the yield of Fidaxomycin reached 320mg/l.

Example 24

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, pH adjustment to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculum amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 9 days. Other conditions were the same as the implementation Example 1, the yield of Fidaxomycin reached 345mg/l.

Example 25

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, pH adjusted to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 10 days. Other conditions were the same as the implementation Example 1, the yield of fidaxomicin reached 648mg/l.

Example 26

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, pH adjustment to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 11 days. Other conditions were the same as the implementation Example 1, the yield of fidaxomicin reached 835mg/l.

Example 27

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, pH adjusted to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 13 days. Other conditions were the same as the implementation Example 1, the yield of fidaxomicin reached 902mg/l.

Example 28

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, pH adjusted to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 14 days. Other conditions were the same as the implementation Example 1, the yield of Fidaxomycin reached 1050mg/l.

Example 29

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, pH adjustment to 7.3.

The seed solution prepared according to Example 1 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium), the fermentation temperature was 28°C, and the fermentation period was 15 days. Other conditions were the same as the implementation Example 1, the yield of fidaxomicin reached 782mg/l.

Example 30

Take a strain of Dactylosporangium aurantiacum NRRL18085 and inoculate it on the slant medium, cultivate it at 30°C for 4 days and activate it, then inoculate it on the seed medium. Bed culture for 48 hours to get a first-class seed solution. The primary seed liquid was inoculated with 5% (percentage is the volume percentage relative to the total volume of the seed medium) in 750ml seed medium with a liquid volume of 100ml, and cultured on a shaker at 28°C for 48 hours to obtain the secondary seed liquid. The secondary seed liquid was inoculated into a 5L fermenter with a liquid volume of 2L at an inoculum amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium) for fermentation. The fermentation temperature was 28°C, and the fermentation period was 10 days. During the process, the dissolved oxygen was controlled at 20% (the percentage is the volume percentage relative to the total volume of the fermentation medium). After the fermentation, three times the volume of methanol was added to the fermentation medium, the supernatant was obtained by centrifugation after 30 minutes of ultrasonication, and the yield of fidaxomicin was determined to be 1222 mg/l by HPLC.

The raw material components of the slant medium used include 0.4% of yeast extract powder, 1% of malt extract powder, 0.4% of glucose and 2.0% of agar, and the pH is adjusted to 7.2.

The raw material components of the seed medium used include 0.1% glucose, 2.4% starch, 0.5% yeast extract powder, 0.5% tryptone, 0.3% beef extract and 0.4% CaCO ₃ , and adjust the pH to 7.3.

The raw material components of the fermentation medium include maltose 6.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ 7H ₂ O 0.2%, bubble enemy 0.1%, adjust the pH to 7.3.

Example 31

The raw material components of the fermentation medium include maltose 7.5%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ ·7H ₂ O 0.2%, bubble enemy 0.1%, adjust the pH to 7.3.

The secondary seed liquid prepared according to Example 30 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium) to carry out fermentation in a 5L fermenter at a fermentation temperature of 28°C and a fermentation cycle of 10 Day, controlled dissolved oxygen 20% in the fermentation process (percentage is volume percent relative to the total volume of fermentation medium), other conditions are the same as embodiment 30, the output of fidaxomicin reaches 1684mg/l.

Example 32

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ ·7H ₂ O 0.2%, bubble enemy 0.1%, adjust the pH to 7.3.

The secondary seed liquid prepared according to Example 30 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium) to carry out fermentation in a 5L fermenter at a fermentation temperature of 28°C and a fermentation cycle of 10 Day, controlled dissolved oxygen 20% in the fermentation process (percentage is volume percent relative to the total volume of fermentation medium), other conditions are the same as embodiment 30, the output of fidaxomicin reaches 1897mg/l.

Example 33

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ ·7H ₂ O 0.2%, bubble enemy 0.1%, adjust the pH to 7.3.

The secondary seed liquid prepared according to Example 30 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium) to carry out fermentation in a 5L fermenter at a fermentation temperature of 28°C and a fermentation cycle of 10 Day, control dissolved oxygen 30% in the fermentation process (percentage is volume percent relative to the total volume of fermentation medium), other conditions are the same as embodiment 30, the output of fidaxomicin reaches 2219mg/l.

Comparative Example 1

The raw material components of the fermentation medium include maltose 9.0%, glycerin 2.0%, cottonseed cake powder 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO ₃ 0.4% and MgSO ₄ ·7H ₂ O 0.2%, bubble enemy 0.1%, adjust the pH to 7.3.

The secondary seed liquid prepared according to Example 30 was inoculated in the fermentation medium with an inoculation amount of 10% (percentage is the volume percentage relative to the total volume of the fermentation medium) to carry out fermentation in a 5L fermenter at a fermentation temperature of 28°C and a fermentation cycle of 10 Day, control dissolved oxygen 0.5% in the fermentation process (percentage is volume percent relative to the total volume of fermentation medium), other conditions are the same as embodiment 30, the output of fidaxomicin reaches 175mg/l.

It should be understood that after reading the above content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (14)

1. a kind of fermentation medium with dactylosporangium aurantiacum fermenting and producing antibiotic feldamycin, it is characterised in that：Its raw material Component include maltose 9.0%, glycerine 3.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, dusty yeast 1.0%, CaCO₃0.4% and MgSO₄·7H₂O0.2%, pH 7.3；Percentage is the quality percentage relative to fermentation medium gross mass Than.

2. a kind of fermentation medium with dactylosporangium aurantiacum fermenting and producing antibiotic feldamycin, it is characterised in that：Its raw material Component include maltose 9.0%, glycerine 4.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, dusty yeast 1.0%, CaCO₃0.4% and MgSO₄·7H₂O0.2%, pH 7.3；Percentage is the quality percentage relative to fermentation medium gross mass Than.

3. a kind of fermentation process with dactylosporangium aurantiacum fermenting and producing antibiotic feldamycin, it is characterised in that including following step Suddenly：Seed culture medium is inoculated in after dactylosporangium aurantiacum is activated and obtains seed liquor, seed liquor is seeded in such as claims 1 or 2 In the fermentation medium, fermented and cultured, you can.

4. fermentation process as claimed in claim 3, it is characterised in that：Inoculation during the seed liquor inoculation of the fermented and cultured It is 5~25% to measure, and percentage is the percent by volume relative to fermentation medium total volume.

5. fermentation process as claimed in claim 3, it is characterised in that：Inoculation during the seed liquor inoculation of the fermented and cultured It is 5~15% to measure, and percentage is the percent by volume relative to fermentation medium total volume.

6. fermentation process as claimed in claim 3, it is characterised in that：The fermentation temperature of the fermented and cultured is 25~32 ℃；The fermentation period of the fermented and cultured is 8~15 days.

7. fermentation process as claimed in claim 3, it is characterised in that：The fermentation temperature of the fermented and cultured is 27~32 ℃；The fermentation period of the fermented and cultured is 10~15 days.

8. a kind of fermentation process with dactylosporangium aurantiacum fermenting and producing antibiotic feldamycin, it is characterised in that including following step Suddenly：Seed culture medium is inoculated in after dactylosporangium aurantiacum is activated and obtains primary seed solution, primary seed solution is inoculated in seed culture Secondary seed solution is obtained after base, secondary seed solution is seeded in fermentation medium as claimed in claim 1 or 2, controls dissolved oxygen Be 1~30%, percentage be relative to fermentation medium total volume percent by volume, fermented and cultured, you can.

9. fermentation process as claimed in claim 8, it is characterised in that：During the secondary seed solution inoculation of the fermented and cultured Inoculum concentration is 5~25%, and percentage is the percent by volume relative to fermentation medium total volume.

10. fermentation process as claimed in claim 8, it is characterised in that：During the secondary seed solution inoculation of the fermented and cultured Inoculum concentration for 5~15%, percentage is the percent by volume relative to fermentation medium total volume.

11. fermentation process as claimed in claim 8, it is characterised in that：The fermentation temperature of the fermented and cultured is 25~32 ℃；The fermentation period of the fermented and cultured is 8~15 days.

12. fermentation process as claimed in claim 8, it is characterised in that：The fermentation temperature of the fermented and cultured is 27~32 ℃；The fermentation period of the fermented and cultured is 10~15 days.

13. fermentation process as claimed in claim 8, it is characterised in that：The control dissolved oxygen of the fermented and cultured for 10~ 30%, percentage is the percent by volume relative to fermentation medium total volume.

14. fermentation process as claimed in claim 8, it is characterised in that：The control dissolved oxygen of the fermented and cultured for 20~ 30%, percentage is the percent by volume relative to fermentation medium total volume.